Pertanika J. Sci. & Techno!. 2(1): 47-53 (1994)

ISSN: 0128-7680

© Universiti Pertanian Malaysia Press

A Spectrophotometric Method for the Determination ofGlucose with Glucose Oxidase [E.C. 1.11.1.7] UsingTitanium (IV)-4-(2'-pyridylazo)Resorcinol Reagent

Kamaruzaman Ampon, Wan Zin Wan Yunus1

and Abu Bakar SallehDepartment ofBiochemistry and Microbiology,

1Department of Chemistry,Faculty ofScience and Environmental Studies,

Universiti Pmanian Malaysia43400 UPM Serdang, Selangar, Malaysia.

Received 13July 1993

ABSTRAK

Satu campuran Ti(IV) dan 4-(2-piridilazo)resorsinol (Ti-PAR) didapati bergunauntuk mengesan hidrogen peroksida dalam amaun yang sedikit secaraspektrofotometri. Kompleksyang terbentukmenyerap pada508 nm dan keserapantersebut berkadar dengan kepekatan H 20 2 yang ditambah. Reagen Ti-PAR initelah digunakan di dalam pengasaian glukosa melalui pengkupelannya denganenzim glukosa oksidase yang menghasilkan H 20 2• Kaedah penentuan glukosabegini adalah cepatdan mudah dan tidak dipengaruh oleh bahan-bahan terturun.

ABSTRACT

A mixture of Ti(IV) and 4-(2-pyridylazo) resorcinol was found to be useful in thespectrophotometric determination of trace amounts of hydrogen peroxide. Theabsorbance of the complex formed at 508 nm was proportional to the concentra­tion ofhydrogen peroxide added. The reagent was applied to the assay ofglucosethrough coupling with glucose oxidase which produces HP2' This method ofglucose determination was rapid, convenient and showed minimal interferencefrom reducible substances.

Keywords: spectrophotometric determination, hydrogen peroxide, Ti-PAR rea­gent, glucose oxidase

INTRODUCTION

Matsubara and Takamura (1980) showed that a mixture of Ti (IV) and 4-(2'­pyridylazo) resorcinol (PAR) (denoted as Ti-PAR) can be used for the deter­mination of trace amounts of hydrogen peroxide (H

20

2). The usual

spectrophotometric procedure for the determination of generated H20 2

(Chibome and Fridovich 1979; Josephy et al. 1982) uses a chromogen in thepresence of a peroxidase to yield a chromophore as shown below.

H20

2+ Chromogen - - - -> Chromophore + H 20

Peroxidase

Kamaruzarnan Ampon, Wan Zin Wan Yunus and Abu Balear Salleh

Some foreign substances, however, can interfere with this detennination.For example, serum which contains reducible substances such as bilirubin,ascorbic acid, uric acid, coenzyrne-A and drug metabolites may influence thereaction either by competing with the chromogen for H20 2

or by reducing thechromophore. Analysis of H20 2 using Ti-PAR reagent, however, was notaffected by the presence of reducible substances and its use in the colorimetricdetennination of trace amounts of H

20

2in biological fluids requires no

peroxidase coupling step.The present paper is concerned with the application ofTi-PAR reagent in

the assay of H20 2produced from the reaction of glucose oxidase on glucose.

MATERIALS AND METHODS

MaterialsTi(IV), PAR and 3,5,3',5'-tetramethylbenzidine (TMB) were obtained fromSigma Chemical Co. Glucose oxidase (GOD) and horseradish peroxidase werealso purchased from Sigma. Hydrogen peroxide (30%) was obtained fromFisher Scientific. All chemicals used were ofanalytical reagent grade and wereused without further purification.

The solutions of Ti(IV), 4-(2-pyridylazo)resorcinol (PAR) and Ti-PARreagents were prepared as follows: The solution of Ti (IV) was prepared bydissolving titanium tetrachloride (24 ml) in 4M HCl (500 ml), and wasstandardized by titration with ethylenediaminetetra acetic acid (EDTA), fol­lowed by dilution with water to give 1 mM stock titanium (IV) solution. Thesolution ofPARwas prepared bydissolving 22 mg ofPARin 5 ml ofO.2M sodiumhydroxide and diluting to 100 ml with water to give a stock PAR solution (1mM). The Ti-PAR reagent was prepared by mixing 40 ml of the stock Ti(IV)solution with 40 ml of the stock PAR solution. This solution was diluted withwater to 100 ml.

A stock solution of TMB (1 mg/ml) was prepared by dissolving thecompound in 1 M sodium acetate buffer, pH 4.5. A solution ofH20 2

(O.lM) wasprepared as follows: 30% H 20 2 (5.5 ml) was diluted with water to 500 ml andstandardized by titration with potassium pennanganate.

Hydrogen Peroxide DeterminationThe oxidation of TMB by H20 2 through a peroxidase-coupled system, meas­uredat660 nm was used to quantify the presenceofH

20

2(Josephy etal.l982).

Determination using Ti-PAR reagent was according to that described byMatsubara et al. (1985).

Determination ofGlucose Oxidase ActivityThe glucose oxidase activity was determined by two different methods and theresults were compared. In the first method the reaction velocity was deter­mined by an increase in absorbance at 660 nm resulting from the oxidation ofTMB through a peroxidase-coupled system as described by Josephy et al.

48 Pertanika J. Sci. & Techno!. Vo!. 2. No. I, 1994

A Spectrophotometric Method for the Determination of Glucose with Glucose Oxidase

(1982). One unit of enzyme activity (U) was defined as the amount of GODwhich will oxidize 1.0 pmole of ~-D-glucose to D-gluconic acid and HP2 permin under the conditions used.

In the second method the H 20 2 generated from the reaction of glucoseoxidase and glucose was analysed using the Ti-PAR reagent. The effects ofpH,temperature, reaction time, concentration of glucose oxidase and glucoseused were studied in preliminary experimen ts in order to optimize the reactionconditions for the determination ofH20 2using Ti-PARreagent. The followinggeneral conditions were used: To a mixture of0.5 ml glucose solution (O.lmMin Tris buffer pH 7.0, 100 mM) and 0.4 ml Ti-PAR reagent previously equili­brated at 37°Cwas added 5 -1 0 III glucose oxidase (100 U/ ml) .The mixture wasincubated for 15 min, cooled to room temperature and the absorbancemeasured at 508 nm. For the control assay, a solution containing the samecomponents as above was prepared, except that heat-denatured GOD wasadded. All the measurements were made with reference to a reagent blank.

RESULTS

A comparison of the H 20 2 determinations using Ti-PAR reagent with aperoxidase-coupledsystem is given inFig. 1. The sensitivity ofthe determinationsis comparable.

0.6

Q)()<::CIl

0.4-e0

'".0<{

0~2

5 10 15 20

Hydrogen peroxide (11M)

Fig. 1. Standard curve for Hp2 under standard assay conditions determined usingthe Ti-PAR system (absorbance at 508 nm, .-) and the oxidation of TMB through a peroxidase­

coupled system (absorbance at 660 nm, -0-). Each point is the mean of three determinations

In order to select the best conditions for the combined use of the Ti-PARreagent and glucose oxidase in the determination of H 20 2 and glucose, acareful consideration of the incubation time, pH and enzyme concentration

Pertanika J. Sci. & Technol. Vol. 2. No. I, 1994 49

Kamaruzaman Ampon, Wan Zin Wan Yunus and Abu Bakar Salleh

was made. The optimal pH for the glucose oxidase catalysed reaction wasreported to be about 6.0 (Bright and Appleby 1969) while the optimal colourformation of the Ti-PARreagentwas between pH 7.6 and 12.0 (Matsubara etal.1985). The effect of different levels of pH on the absorbance was thusinvestigated by examining the enzymatic and colour formation processes. Theabsorbance response was found to be fairly constant in the pH range of 6-7.5(Fig. 2). Thus, to compromise on the optimal enzym~ reaction and colourformation, the pH of the solution was maintained at 7.0 throughout allenzymatic and chromogenic reactions.

0.4 A__0

E ~cco 0.30!!2.

~BOJuc

0.2ell-e0(fJ.c<l: 0.1

6 7 8

pH

9 10

Fig. 2. Effects ofpH on the absorbance of the Ti-PAR-HP2 system (A) and the Ti-PARreagent combined with glucose oxidase system (B) under standard assay conditions

In order to determine the appropriate glucose oxidase concentration, theeffect of enzyme activity on the absorbance was examined. Relatively constantabsorbance values were obtained for GOD activity values above 0.5 U afterincubation for 15 min at 37°C with glucose (Fig. 3).

A plot ofabsorbance against glucose concentration in a GOD-coupled Ti­PARsystem (GOD = 0.5 V/ml solution) gave a linear relationship (Fig. 4). Themethod may thus be applicable for the determination ofglucose, especially inthe lower concentration range of up to about 20 IlM.

The effects of certain foreign substances, such as the anticoagulant EDTAand ascorbic acid, added to the test solution were also examined. Addition ofascorbic acid (10 mg/l) and EDTA (200 mg/l) (results not shown) had nosignificant effects on the glucose assay (Fig. 4A). On the other hand, thepresence of ascorbic acid in the peroxidase-TMB system significantly reducedthe absorbance (Fig. 4B).

50 Pertanika J. Sci. & Techno!. Vo!. 2. No. I, 1994

A Spectrophotometric Method for the Determination of Glucose with Glucose Oxidase

0.2 .----. •c ---.....c<0 I0~G>Uc

I·eu 0.1 -n(;<Jl

In«

I·0.5 1.0 1.5

Glucose oxidase (U/3ml)

Fig. 3. Effects ofglucose oxidase concentration on the absorbance under standard

assay conditions. Each point is the mean of two determinations

+ Ascorbic acid

0.5 A 1.0E Ec cOJ 0.4 0 0 .8a~ <0OJ <0u 0.3 -;; 0.6cco Q.Da c

'" 0.2 2 0.4.D<l: ....

0

o. , .2 0.2«:

4 6 8 10 12 4 6 8 10 12

Glucose (~IM)

Fig. 4. Standard curve for glucose under standard assay conditions using Ti-PARreagent (A) and peroxidase-coupled system (B) £n the presence and absence ofascorbic

acid. Each point is the mean of three determinations

In addition, the GOD-coupled Ti-PAR method was also applied to themeasurement of glucose in solutions which were spiked with known amountsof glucose. The determinations were compared with the peroxidase-coupledmethod (Fig. 5). A good correlation was observed for the parameters underanalysis (r=0.878).

Pertanika J. Sci. & Technol. Vol. 2. No. I, 1994 51

Kalllaruzalllan Alllpon. Wan Zin Wan Yunus and Abu Bakar Salleh

•• ••10 •~~ 8"'C0~ 6-Q) •E •a: 4 •0: •F 2

2 4 6 8 10 12

Peroxidase method (11M)Fig. 5. Correlation ofglucose concentrations as determined by the Ti-PAR method

with that determined by the peroxidase-coupled method

DISCUSSION

It was previously reported that the Ti-PARreagent could be used to detect traceamoun ts of hydrogen peroxide (Matsubara et at. 1983). Thus Ti-PAR reagentwas used in this experiment for the determination of free hydrogen peroxideby glucose oxidase reaction. The method was rapid, convenient and reproduc­ible compared to using a chromogen in the presence of peroxidase.

As reported previously (Matsubara and Takamura 1980) and confirmed inthe presentwork, the absorbance spectrum ofthe Ti-PAR-H

20

2complex shows

a peak in the pH range between 7.6 and 12.0. The absorbance at 508 nm wasproportional to the concentration of added H 20 2, ranging from 0.5 to 30 IlM(Fig. 1) with a molar absorption coefficient (E) of about 3.6 x 104 M· l em-I.Constant absorption values were obtained within a few minutes following theaddition of hydrogen peroxide to the Ti-PAR reagent and remained un­changed over 24 h at room temperature. Interference which was caused by thepresence of excess Ti-PAR can be minimized by incubation at 37°C for a fewminutes.

The results obtained in this research show that the Ti-PAR reagent can beused to assay free hydrogen peroxide and glucose and has an advantage overother methods. It does not require the use of peroxidase in the determination.The presence of peroxidase is known to prompt the oxidation of chromogensand other reducible substances. Thus, the Ti-PAR method is not affected by thepresence of reducible substances, such as ascorbic acid. The sensitivity of theTi-PAR reagent is also quite high. These findings and those reported byMatsubara et al. (1983) strongly support the use of this reagent for thecolorimetric determination of trace amounts ofhydrogen peroxide in biologi­cal fluid.

The Ti-PAR reagent, thus has been successfully used to determine tracebiological substances in serum using appropriate enzyme combinations toproduce H

20

2through enzymatic oxidation. Matsubara and Takamura (1980)

52 Pcrtanika J. Sci. & Techno!. Vo!. 2. No.1. 1994

A Spectrophotometric Method for the Determination of Glucose with Glucose Oxidase

used the Ti-PAR reagent in combination with glucose oxidase, uricase andcholesterol oxidase for the determination of glucose, urate and cholesterol,respectively, in human serum. Similarly, Matsubara et al. (1983) used a combi­nation of acyl-CoA synthase and acyl-CoA oxidase to assay for free fatty acid inserum.

ACKNOWLEDGEMENTS

This projectwas financed by the Ministry ofScience, Technology and Environ­ment, Malaysia (IRPA Project No. 4--07-05-042).

REFERENCESBRIGHT, HJ. and M. APPLEBY. 1969. The pH dependence of the individual steps in the glu­

cose oxidase reaction.] Biol. Chem. 244: 3625-3630.

CLAIBORNE, A. and 1. FRIDOVICH. 1979. Chemical and enzymatic intermediates in the peroxida­tion of o-dianisidine by horseradishperoxidase. Spectral properties of the productsof o-dianisidine oxidation. Biochemistry 18: 2324-2329.

]OSEPHY, P.D., T. ELING and R.P. MAsON. 1982. The horseradish peroxidase-eatalysed oxida­tion of3,5,3',5'-tetramethylbenzidine. Free radical and charge-transfer complex inter­mediates.] Biol. Chem. 277: 3669-3675.

MATSUBARA, C., T. IWAMOTO, Y. NISHIKAWA and K. TAKAMURA.1985. Coloured species formedfrom titanium (IV)-4-(2'- pyridylazo) resorcinol reagents in the spectrophotometricdetermination of trace amounts of hydrogen peroxide.] Chem. Soc. Dalton Trans. 1:81-84.

MATSUBARA, C., Y. NISHIKAWA, Y. YOSHIDA and K. TAKAMURA. 1983. Aspectrophotometricmethod for the determination of free fatty acid in serum using acyl-eoenzyme Asynthetase and acyl-eoenzyme A oxidase. Anal. Biochem. 130: 128-133.

MATSUBARA, C. and K. TAKAMURA. 1980. Spectrophotometric determination of traces ofHP2 by the Ti(IV)-PAR and V(v)-XO reagents. Application to the assay for glucose,urate and cholesterol in serum. Bunseki Kagaku 29: 759-764.

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